3.1. Anabaena flos-aquae UTEX 1444: Strain, Cultivation and Extraction

The genus Anabaena BORY ex BORN. et FLAH. 1888 encompasses filamentous, non-branched species, which differ in the form of cells, coiling of trichomes, shape and size of akinetes [26]. Anabaena species exhibit a wide range of strain-specific variability, and environmental conditions mainly affect cell dimensions and size of heterocysts [27].

The species A. flos-aquae Brébisson ex Bornet et Flahault 1888, is a free-floating organism, whose coiled filaments can be solitary or irregularly assembled, with narrow mucilaginous sheath. Cells are usually spherical to barrel-shaped, whereas akinetes can be cylindrical or elliptical and heterocysts usually spherical [28]. A. flos-aquae is diffused in freshwater environments worldwide and is responsible of blooms that can have noxious effects on other organisms, due to the excretion of toxic metabolites, as anatoxins [29].

From a taxonomical point of view, A. flos-aquae is a combined morphological group, in that an alternation of regularly and irregularly coiled filaments is observed for the same strain in laboratory cultures; moreover, vegetative cell shape and size, along with heterocysts and akinetes dimensions, were largely variable among different populations [30]. Considering that the type species of the genus Anabaena, A. oscillarioides Bory ex Bornet et Flahault 1888 is a benthic type, Wacklin et al. [31] proposed to transfer the planktic Anabaena types under the genus Dolichospermum, designating A. flos-aquae as the type species of the genus.

The strain Anabaena flos-aquae UTEX 1444 was grown in BG-11 medium containing (per liter): 2.21 mg of Na₂EDTA, 12 mg of Citric Acid, 1 g of NaNO₃, 80 mg K₂HPO₄·3H₂O, 150 mg of MgSO₄·7H₂O, 52.8 mg of CaCl₂, 34.2 mg of NaCO₃, 2 mL of Nitsch solution (H₂SO₄, MnSO₄·H₂O, ZnSO₄·7H₂O, CuSO₄·5H₂O, H₃BO₃, Na₂MoO₄·2H₂O, CoCl₃·6H₂O), 500 µL of NiSO₄ (NH₄)₂ SO₄·6H₂O (0.1 mM stock), 200 µL of Na₂SeO₄ (0.1 mM stock). Cultures were grown in six different concentrations of iron supplemented as ferric ammonium citrate in BG11 medium; serial dilutions from a mother ferric ammonium citrate solution (100 µM) were prepared in order to get the following final concentrations: 0 µM Fe³⁺ (#1), 5 µM Fe³⁺ (#2), 10 µM Fe³⁺ (#3), 20 µM Fe³⁺ (#4), 60 µM Fe³⁺ (#5), and 100 µM Fe³⁺ (#6). All cultures were made in triplicates and were incubated at room temperature (18–25 °C) for 30 days. Cultures were observed under an optical microscope (OPTECH, Biostar B3) and a fluorescence microscope (iRiS Digital Cell Imaging System–Logos Biosystems, Boston, MA, USA); then, they were vortexed, sonicated (ARGO Lab Digital Ultrasonic Cleaner, Model-DU 32, Carpi, Italy) at 20 KHz for 10 min at 23 °C, and centrifuged (HERMLE Labortechnik, Model-Z36HK, Wehingen, Germany) at 10,000 rpm for 5 min at 25 °C to separate the solid pellets from the liquid supernatant. Pellets were extracted with organic solvents as follows: MeOH (100%, 0.3 L), MeOH/CHCl₃ (1:1, 0.3 L), and CHCl₃ (100%, 0.3 L). All the extracts were paper filtered and concentrated under vacuum, yielding (average weights): 9.6 mg (#1), 6.6 mg (#2), 6.3 mg (#3), 9.0 mg (#4), 7.2 mg (#5) and 9.3 mg (#6) of MeOH extract; 2.3 mg (#1), 3.5 mg (#2), 4.3 mg (#3), 3.4 mg (#4), 1.2 mg (#5) and 2.5 mg (#6) of MeOH/CHCl₃ extract; 7.6 mg (#1), 0.5 mg (#2), 1.7 mg (#3), 0.6 mg (#4), 1.3 mg (#5) and 0.3 mg (#6) of the CHCl₃ extracts. Supernatants were extracted with BuOH. BuOH phases were concentrated under vacuum affording to 3.5 mg (#1), 13.3 mg (#2), 30.3 mg (#3), 45.6 mg (#4), 40.3 mg (#5) and 40.6 mg (#6) of extract. Each extract was resuspended in MeOH (100%, 5 mg/mL) for the subsequent LC-HRMS analyses.